The harmonious development of the human embryo is essential if individuals are to meet successfully the demands of several crucial life steps on (i.e., birth). Alterations of the normal developmental patterns can cause a wide variety of health problems ranging from undetectable or mild changes to severe malformations, even lethality. Therefore, it becomes critical to know how development occurs normally if we are ever going to be able to design rational preventive or therapeutic measures. The Hox genes have been implicated strongly in several aspects of normal development, and this proposal deals with experiments designed to study their normal role in vivo through the use of mouse mutants generated by gene targeting in embryonic stem (ES) cells. The long-term goal of this proposal is to carry out an elaborate genetic dissection of part of one of the mammalian Hox clusters, HoxB. The initial approach is to scan genetically the 5' end of the cluster (Hoxb-9, Hoxb, and Hoxb-7 genes) through the production and analysis of a series of overlapping deletion mutants (Specific Aim 1). Then, analysis of the phenotypic consequences of the different deletions will guide further molecular and genetic characterization (i.e., identification of new cDNAs and regulatory regions, generation of additional mouse mutants). Specific Aims 2 and 3 will deal with a focused in vivo analysis of the role of the most 5' gene of the HoxB cluster, Hoxb-9. Specific Aim 2 will describe the analysis of the coding region of the gene, and Specific Aim 3 will study the regulatory elements responsible for its proper expression during development. These experiments will involve gene targeting to generate mouse mutants, transgenic mice with lacZ reporter genes, and in vitro DNA- protein binding studies. All the in vivo work will include detailed phenotypic characterization (i.e., morphological and molecular criteria) with the intention of finding opportunities to develop assays to study the basic cellular mechanisms of Hox gene action.